The long term goal of the laboratory is to understand mechanisms of regulation of contraction in muscle and non-muscle cells. Striated muscle thin filament regulation involving troponin and tropomyosin is of particular interest. Experiments are outlined in the present proposal to use site-directed mutagenesis for study of structure-function relationships in troponin C. Troponin C binds calcium and regulates contraction via interaction with troponin I and troponin T. Crystallographic analysis of the structure has shown that troponin C, like calmodulin, has two calcium binding domains connected by a long central helix. The structural features of the protein that are important for its function as a calcium binding regulatory protein and transmission of information about the state of calcium binding in the regulatory domain to other components of the thin filament will be investigated. A cDNA encoding avian troponin C has been constructed from oligonucleotides and the protein has been expressed and purified from E. coli. The functional role of the long helix connecting the two domains of troponin C, a conserved structure in calcium regulatory proteins, will be evaluated by altering its structure in a variety of ways: its helicity and stability, its length and orientation of the domains relative to each other. Oligonucleotide directed mutagenesis will be used to change individual residues and to make deletions and insertions. The mutants will be analyzed for calcium binding, complex formation with other troponin components, regulation of the actomyosin ATPase, and ultimately substitution in skinned fibers and X-ray crystallography. An additional aim of the experiments is to contribute to our general understanding of the requirements for alpha-helical structure in proteins. The second specific aim is to determine the location and structural requirements of the troponin I and troponin T binding sites on-troponin C. We will use two approaches: construction of troponin C-calmodulin chimeras and mutagenesis of residues on the surface of the protein that do not contribute to the hydrophobic core of the protein. Analysis of chimeras should indicate which residues are critical for troponin C specific functions. Mutagenesis of specific residues will follow.